The link between electrical excitation and contraction of a skeletal muscle cell (e-c coupling) involves signaling between two calcium channels: the dihydropyridine receptor (Oqs-DHPR) which is a voltage-gated Ca 2+ channel in the plasma membrane and ryanodine receptor type 1 (RyR1) which is a Ca 2+ release channel in the sarcoplasmic reticulum (SR). The long-term objective of this proposal is to understand the molecular basis for signaling between RyR1 and alpha1s- DHPR. The basic experimental approach will be to analyze the functional consequences of expressing cDNAs encoding DHPRs or RyRs in cultured muscle cells (myotubes) obtained from animals genetically null for one or more muscle proteins: dysgenic (alpha1s-DHPR null), dyspedic (RyR1 null), double knockout (null for both alpha1s DHPR and RyR1) and homerless (null for homer 1,2 and 3). The proposal has four specific aims. The first aim is to quantify "retrograde" signaling, whereby the RyR regulates entry of extracellular Ca 2+, for chimeric and mutant RyRs and to probe the functional importance of this entry. The second aim is to characterize the role of the accessory proteins homer and calmodulin in excitation-contraction coupling and in retrograde signaling. The third aim is to determine whether fluorescence energy resonance transfer (FRET) occurs between fluorescent protein-tagged alpha1-DHPRs assembled into plasma membranes at junctions with the SR and if it does to determine whether the FRET efficiency is affected by pharmacological manipulations of RyR1. The fourth aim is to explore interactions between DHPRs and RyRs in double knock-out (null for both alpha1s DHPR and RyR1), including a determination of whether skeletal-type e-c coupling can occur after expression in double knock-out myotubes of (alpha1c-DHPR and RyR2, the DHPR and RyR isoforms predominant in cardiac muscle. In addition to providing new insights into a basic muscle function, the proposed experiments will also provide knowledge essential for understanding the inherited human muscle diseases hypokalemic periodic paralysis (caused by mutations of alpha1s-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1).